Hemoglobin-Inorganic Hybrid Nanoflowers: Synthesis And Applications For Carbene Reaction

As a kind of non-toxic and environmentally friendly biocatalysts,enzymes have been widely used in organic synthesis because of their high catalytic performance,mild reaction conditions and strong specificity.Because free enzymes are difficult to recover and reuse,strict requirements on reaction conditions,easy inactivation and other inherent limitations,the practical application in industrial production of enzymes is limited.The immobilization of enzyme molecules in the carrier matrix can overcome the limitations of enzyme effectively through appropriate immobilization carrier and immobilization method,so as to promote the wider application of enzyme catalysis in industrial production field.The discovery that enzymes can catalyze many unnatural reactions,a phenomenon known as enzyme catalytic promiscuity,has greatly expanded the use of enzymes in organic synthesis.Hemoproteins are a class of proteins containing iron porphyrins（hemoglobin,myoglobin,cytochrome,peroxidase and so on）that have the ability to catalyze a variety of unnatural reactions,such as oxidation,carbene transfer,and carbene X-H insertion.These studies opened up a new world of types of organic reactions catalyzed by heme proteins.Our research group has previously reported that hemoglobin（Hb）can efficiently catalyze the synthesis of quinoxaline compounds by carbene reaction,but free Hb is difficult to recycle and has poor protein stability.To solve the above problems,we prepared hemoglobin-copper phosphate hybrid nanoflowers（Hb-Cu₃（PO₄）₂HNFs）by co-incubating bovine hemoglobin with copper ions and used it to catalyze carbene reaction to synthesize quinoxalines.The results show that Hb-Cu₃（PO₄）₂HNFs show excellent catalytic performance in this reaction,and Hb-Cu₃（PO₄）₂HNFs can still get up to 85%yield after repeated 10 times,showing excellent reuse performance.In addition,many studies have proved that hemoproteins can catalyze carbene N-H insertion reaction to construct C-N bond,which provides a simple way for the synthesis ofα-amino acid derivatives,α-amino ketone and nitrogen heterocyclic rings.Carbene N-H insertion with aniline and ethyl diazoacetate（EDA）as substrates is a model for evaluating the activity of carbene insertion catalyzed by hemoproteins,but the carbene N-H insertion catalyzed by hemoglobin has not been explored.In this paper,Hb-Cu₃（PO₄）₂HNFs were used to catalyze carbene N-H insertion reaction between aniline and ethyl diazoacetate,to explore the catalytic performance and reusability of the immobilized enzyme.In this study,we optimized the reaction conditions from three aspects of reaction time,reaction temperature and catalyst dosage by controlling single factor variables.Under optimal conditions,aniline（1mmol）,ethyl diazoacetate（1 mmol）,deionized water（10 m L）,Hb-Cu₃（PO₄）₂HNFs（protein content:15 mg）,sodium hydrosulfite（25 mg）,25°C,12 h,Hb-Cu₃（PO₄）₂HNF catalyzed carbene N-H insertion reaction yields up to 87%,and free Hb yields up to 74%.Furthermore,after 15 repetitions,the yield of Hb-Cu₃（PO₄）₂HNF was still up to 75%of the initial yield,indicating that the immobilized Hb-Cu₃（PO₄）₂HNF had excellent reusability performance.The work in this paper broadens the application of unnatural reactions catalyzed by hemoglobin.